JP6000671B2 - Photodetection system and microscope system - Google Patents

Description

  The present invention relates to a light detection system and a microscope system.

  Conventionally, in the biological research field, particularly in the research market using a microscope, there is an increasing demand for observing a faster dynamic reaction of a living body. In the microscope, when the frame rate is increased, the detection time required for acquiring one frame image is shortened. Therefore, the detector is required to detect weaker light. For example, a photon counting (photon measurement) technique is used.

  In addition, if the exposure time (for example, scanning speed in a laser scanning microscope) is changed under the same light incident conditions, the number of photons incident on one pixel changes. The image brightness changes. Therefore, each time the user changes the scanning speed, the user needs to change observation conditions (HV (HIGH VOLTAGE, analog gain in a broad sense) and laser light quantity), which is bothersome.

  On the other hand, the scanning laser microscope described in Patent Document 1 changes the HV according to the exposure time or changes the amount of light incident on the detector, thereby changing the GUI regardless of the scanning speed. The luminance value displayed in (Graphical User Interface) is made constant.

JP 2005-215357 A

  However, in the scanning laser microscope described in Patent Document 1, in the case of photon counting, the number of incident photons increases and decreases according to the exposure time as described above, and the image luminance changes. There is an inconvenience that the number of photons obtained does not change only by changing the high value. Also, changing the amount of light incident on the detector increases the amount of light incident on the detector during a short exposure time, which may induce multiphoton events, resulting in lack of luminance linearity. There is an inconvenience of being an image.

  An object of the present invention is to provide a photodetection system and a microscope system that can keep the brightness of an image to be displayed constant even when the exposure time is changed during photon counting detection.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light receiving unit that receives observation light emitted from a sample, photoelectrically converts the observation light received by the light receiving unit, and outputs a current signal having a magnitude corresponding to the amount of light. A photon measurement processing unit that measures the number of photons included in the current signal output from the photoelectric conversion unit, an input unit that inputs an exposure time during which the observation light is exposed in the light receiving unit, and a reference the reciprocal of the ratio of the a actual exposure time actual exposure time input by the input unit with respect to the reference exposure time is a predetermined exposure time, by multiplying the number of photons measured by the photon counting processing unit to obtain An optical construction system comprising an image construction unit for constructing an image of the sample based on a luminance value obtained.

  According to the present invention, when the exposure time of the light receiving unit of the photoelectric conversion unit input by the input unit changes, the number of photons included in the current signal obtained by photoelectrically converting the observation light by the photoelectric conversion unit depends on the exposure time. The photon measurement processing unit measures the number of photons contained in the current signal from the photoelectric conversion unit, and the image construction unit multiplies the photon number by the inverse of the ratio of the actual exposure time to the reference exposure time. Thus, the luminance value for image construction can be made constant regardless of the change in the number of photons. Therefore, by constructing an image of the sample based on this luminance value, an image having a constant brightness can be acquired regardless of the exposure time of the observation light in the light receiving unit of the photoelectric conversion unit.

  In the above invention, the image construction unit includes a plurality of lookup tables in which the number of photons and the luminance value are associated with each other for each exposure time, and from the lookup table selected based on the actual exposure time, The luminance value associated with the number of photons measured by the measurement processing unit may be read out.

  By constructing in this way, it is possible to easily determine the luminance value and quickly construct the sample image by simply preparing a plurality of possible lookup tables in advance, omitting the time required for arithmetic processing. Can do.

Further, in the above-mentioned invention, said image construction unit calculates the number of photons measured by the photon counting processing unit, wherein the actual exposure time, the luminance values by processing the said reference exposure time It is good.
By configuring in this way, it is possible to determine an appropriate luminance value flexibly according to the actual number of photons and exposure time, and to accurately construct an image of the sample.

  The present invention includes any one of the above-described light detection systems, a scanning unit that reflects illumination light emitted from a light source and scans the sample, and irradiates the sample with the illumination light reflected by the scanning unit. An objective lens that condenses the observation light emitted from the sample and received by the light receiving unit of the photoelectric conversion unit, and the illumination light on the sample by the scanning unit as the actual exposure time A microscope system using a scanning time of 1 mm is provided.

  According to the present invention, it is possible to acquire an image in a range in which illumination light is scanned on the sample by the scanning unit. In this case, even if the scanning time of the illumination light on the sample by the scanning unit is changed, the brightness of the sample image can be kept constant before and after the change.

  According to the present invention, when photon counting is detected, the brightness of an image to be displayed can be kept constant even if the exposure time is changed.

1 is a schematic configuration diagram showing a microscope system according to a first embodiment of the present invention. It is a block diagram which shows the photon detection system of FIG. It is a graph which shows the look-up table which linked | related the exposure time which concerns on 1st Embodiment of this invention, the display maximum gradation, and the number of photons. It is a graph which shows the relationship between the number of photons and a display gradation. It is a block diagram which shows the photon detection system which concerns on 2nd Embodiment of this invention. It is a graph which shows the look-up table which linked | related real exposure time, the display maximum gradation, and the number of photons which concern on 2nd Embodiment of this invention. It is a graph which shows the relationship between the number of photons and a display gradation. It is another graph which shows the relationship between the number of photons and a display gradation.

[First Embodiment]
A light detection system and a microscope system according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a microscope system 100 according to this embodiment includes a light source unit 10 that emits laser light, a microscope apparatus 20 that irradiates a sample S with laser light emitted from the light source unit 10, and a microscope apparatus 20. Is provided with a light detection system 40 for detecting fluorescence (observation light) generated in the sample S irradiated with laser light, and a monitor 49 for displaying an image of the sample S for which fluorescence is detected by the light detection system 40. .

  As the light source unit 10, for example, a multi-wavelength laser light source is used. The light source unit 10 includes light sources 11A and 11B such as supercontinuum lasers that generate laser light, a reflection mirror 13 that reflects a laser generated from the light source 11A, and a laser beam that is reflected by the reflection mirror 13. The dichroic mirror (DM) 15 that reflects the laser light generated from the light source 11B and synthesizes the optical path of each of these laser lights, and the acoustooptic device that modulates the laser light whose optical path is synthesized by the dichroic mirror 15 The light control part 17 is provided.

  The light source unit 10 emits laser light from the laser light sources 11A and 11B, and controls the wavelength selection and intensity adjustment by the light control unit 17, thereby emitting laser light having a predetermined intensity in a predetermined wavelength region. It can be done.

  The microscope apparatus 20 includes a microscope main body 30 having a stage 31 on which the sample S is placed, a scanner (scanning unit) 21 that reflects the laser light emitted from the light source unit 10 and scans the sample S, and the scanner 21. A pupil projection lens (PL) 23 that condenses the reflected laser light and a reflection mirror 25 that reflects the laser light condensed by the pupil projection lens 23 toward the microscope main body 30 are provided. As the scanner 21, for example, a galvanometer mirror, a resonant scanner, or an AOD (Acousto-Optic Deflector) can be used.

  In addition to the stage 31, the microscope main body 30 includes an imaging lens (TL) 33 that converts laser light incident on the reflection mirror 25 into parallel light, and laser light converted into parallel light by the imaging lens 33 as a sample. A plurality of objective lenses 35 that irradiate S and collect fluorescence generated in the sample S are provided. Reference numeral 36 denotes a revolver to which an objective lens 35 is attached and a plurality of objective lenses 35 can be alternatively arranged on the optical path of the laser beam.

  The microscope apparatus 20 reflects the laser light from the light source unit 10 to enter the scanner 21, and is condensed by the objective lens 35 to form the imaging lens 33, the reflection mirror 25, the pupil projection lens 23, and the scanner 21. A dichroic mirror (DM) 27 that transmits fluorescence returning in the reverse direction through the laser beam and branches off from the optical path of the laser beam, and a condensing lens (CFL) 29 that collects the fluorescence transmitted through the dichroic mirror 27 A pinhole (PH) 37 that allows a part of the fluorescence condensed by the condenser lens 29 to pass through, and a collimator lens that converts the fluorescence that has passed through the pinhole 37 into parallel light and enters the light detection system 40 ( COL) 39.

  The light detection system 40 detects the fluorescence converted into parallel light by the collimator lens 39 and outputs a current signal, and processes the current signal output from the detector 41. A photodetection circuit 50, a control unit 43 that controls the swing angle of the light control unit 17 and the scanner 21 of the light source unit 10, and a PC (Personal Computer, image construction unit) 45 that constructs an image of the sample S are provided. ing. The light detection system 40 is connected to an input unit 47 that inputs information relating to the exposure time in the detector 41 input by the user to the PC 45 and a monitor 49 that displays an image constructed by the PC 45.

  As the detector 41, for example, a PMT (Photo Multiplier Tube) or a PD (Photo Diode) can be used. The detector 41 includes a light receiving unit 42 that receives fluorescence, photoelectrically converts the fluorescence received by the light receiving unit 42, and outputs a current signal having a magnitude corresponding to the amount of light. .

As shown in FIG. 2, the photodetection circuit 50 is an amplifier (Pre-Amp) 51 that converts the current signal sent from the detector 41 into a voltage signal, and the voltage signal amplified by the amplifier 51 serves as a reference. A comparator (Comparator) 53 that compares a voltage signal (Discrimination Level) and outputs a voltage signal according to the comparison result, and a measuring instrument (Counter) that measures the number of photons contained in the voltage signal output from the comparator 53 , A photon measurement processing unit) 55.
The measuring device 55 is configured to send measurement data (pixel brightness data) of the measured number of photons to the PC 45 via the control unit 43.

The control unit 43 sends the photon count measurement data sent from the measuring instrument 55 to the PC 45.
The input unit 47 includes an exposure time of the light receiving unit 42 input by the user (hereinafter, an actual exposure time input by the user is referred to as “actual exposure time”) and a predetermined exposure time (hereinafter referred to as a reference). The predetermined exposure time becomes “reference exposure time”) is input to the PC 45.

  As shown in FIG. 3, the PC 45 counts the number of photons (incident photons) measured by the measuring device 55 for each actual exposure time (μs) of one pixel in the light receiving unit 42 and the number of photons with respect to the reference exposure time. A look-up table in which a luminance value obtained by multiplying the reciprocal of the ratio of the actual exposure time (in this embodiment, the maximum luminance display gradation) is automatically generated. FIG. 3 illustrates a look-up table when the exposure time (μs) of one pixel is 200 μs, a look-up table when 20 μs, and a look-up table when 2 μs. The same applies to FIG.

  For example, assuming that one photon pulse can be separated at 5 ns, a maximum of 400 counts can be performed in a 2 μs / pixel scan, and thus a look-up table that can display a maximum of 400 gradations is automatically generated by the PC 45. FIG. 4 shows the relationship between the number of incident photons at 200 μs / pixel and the display gradation, the normal relationship between the number of incident photons at 20 μs / pixel and the display gradation, and the incident photon at 20 μs / pixel. The relationship between the number and the display gradation after changing the LUT is shown.

  Further, the PC 45 is provided with a memory (Frame memory) 46 that accumulates the number of photons of each pixel and stores the generated lookup table. The PC 45 selects a lookup table corresponding to the actual exposure time input from the input unit 47 from the memory 46, and displays the maximum display floor associated with the number of photons measured by the measuring instrument 55 from the selected lookup table. The key is read out. Then, the PC 45 constructs an image of the sample S based on the read display maximum gradation. An image constructed by the PC 45 is displayed on the monitor 49.

The operation of the light detection system 40 and the microscope system 100 configured as described above will be described.
In order to acquire an image of the sample S by the microscope system 100 according to the present embodiment, first, the sample S is placed on the stage 31 and laser light having a predetermined intensity is generated from the light source unit 10 in a predetermined wavelength region.

  The laser light emitted from the light source unit 10 is reflected by the dichroic mirror 27, then reflected by the scanner 21, condensed by the pupil projection lens 23, and the objective lens 35 through the reflection mirror 25 and the imaging lens 33. The sample S is irradiated by the above.

  When fluorescence is generated in the sample S by being irradiated with the laser light, the fluorescence is collected by the objective lens 35, and the optical path of the laser light through the imaging lens 33, the reflection mirror 25, the pupil projection lens 23, and the scanner 21. , And passes through the dichroic mirror 27 to be separated from the optical path of the laser beam.

  The fluorescence transmitted through the dichroic mirror 27 is collected by the condenser lens 29, and only the fluorescence generated at the focal position of the objective lens 35 in the sample S passes through the pinhole 37. The fluorescence that has passed through the pinhole 37 is converted into parallel light by the collimator lens 39 and received by the light receiving unit 42 of the detector 41.

  When fluorescence is received by the light receiving unit 42, a current signal having a magnitude corresponding to the amount of light is output from the detector 41, and the amplifier 51 converts the current signal into a voltage signal. The voltage signal is input to the measuring instrument 55 via the comparator 53, and the actual exposure time input from the user to the input unit 47 is also input to the measuring instrument 55 as a pixel clock. The number of photons included in the voltage signal is measured by the measuring device 55 for the exposure time, and is sent to the PC 45 via the control unit 43 as a 1-pixel luminance signal (pixel brightness data).

  In addition, in the input unit 47, the reference exposure time is input by the user, and the reference exposure time and the actual exposure time are input to the PC 45.

  In the PC 45, a lookup table corresponding to the inputted actual exposure time is selected from the memory 46, and the maximum display gradation associated with the number of photons of the measurement data is read from the selected lookup table. The PC 45 constructs an image of the sample S based on the read display maximum gradation and displays it on the monitor 49.

  In this case, when the exposure time of the light receiving unit 42 changes, the number of photons included in the current signal obtained by photoelectrically converting the fluorescence by the detector 41 changes according to the actual exposure time. For each, a lookup table is generated by associating the number of photons contained in the voltage signal and the maximum display gray scale of luminance obtained by multiplying the number of photons by the inverse of the ratio of the actual exposure time to the reference exposure time. Thus, the luminance value for each pixel can be made constant regardless of the change in the number of photons.

  Therefore, by constructing an image of the sample S based on the maximum display gradation read from the lookup table by the PC 45, an image having a constant brightness can be obtained regardless of the fluorescence exposure time in the light receiving unit 42 of the detector 41. Can be acquired. As a result, the user can observe the sample S while viewing an image with a certain brightness displayed on the monitor 49.

  As described above, according to the light detection system 40 and the microscope system 100 according to the present embodiment, when detecting photon counting, the brightness of an image to be displayed is kept constant even if the exposure time is changed, and the sample is displayed with an image having a desired brightness. S can be observed. In addition, by simply preparing a plurality of expected lookup tables in advance, it is possible to easily determine the luminance value by omitting the time required for calculation processing, and to quickly construct an image of the sample S having a certain brightness. Can do.

[Second Embodiment]
Next, a light detection system and a microscope system according to a second embodiment of the present invention will be described.
As shown in FIG. 5, the microscope system 100 according to the present embodiment differs from the first embodiment in the configuration of the light detection system 140.
In the following, portions having the same configuration as those of the light detection system 40 and the microscope system 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  In the light detection system 140, instead of the PC 45, the PC (image construction unit) 145 calculates the number of photons measured by the measuring instrument 55 and the actual exposure time and the reference exposure time of the light receiving unit 42 input by the user. The luminance value is calculated by processing.

  Specifically, the PC 145 calculates the luminance value by multiplying the photon number by the reciprocal of the ratio of the actual exposure time to the reference exposure time. For example, in the case of 1000 counts at 200 μs / pix, 10 counts are obtained at 2 μs / pix, but 100, which is the reciprocal of the ratio of 2 μs / pix (actual exposure time) to 200 μs / pix (reference exposure time), is 10 counts. The luminance value is calculated by multiplying by (number of photons).

  In this embodiment, the PC 145 stores a plurality of lookup tables in which the number of photons and the luminance value (luminance display maximum gradation) are associated with each other for each exposure time as shown in FIG. The lookup table may be referred to together with the arithmetic processing. FIG. 7 shows the relationship between the number of incident photons at 200 μs / pixel and the display gradation, the normal relationship between the number of incident photons at 20 μs / pixel and the display gradation, and the incident photon at 20 μs / pixel. The relationship between the number and the display gradation after the arithmetic processing is shown. FIG. 8 shows the relationship between the number of incident photons at 200 μs / pixel and the display gradation, and the relationship after the arithmetic processing between the number of incident photons at 20 μs / pixel and the display gradation.

The operation of the light detection system 140 and the microscope system 100 configured as described above will be described.
In order to acquire an image of the sample S by the microscope system 100 according to the present embodiment, the sample S is irradiated with laser light, and the fluorescence generated in the sample S is detected by the light receiving unit 42 of the detector 41 as in the first embodiment. Receives light.

  Then, the number of photons contained in the voltage signal obtained by the detector 41 is measured by the measuring device 55, and the measurement data is sent to the PC 145, and the actual exposure of the light receiving unit 42 input by the user through the input unit 47. The time and the reference exposure time are input to the PC 145.

  In the PC 145, the luminance value is calculated by multiplying the number of photons by the reciprocal of the ratio of the actual exposure time to the reference exposure time. Then, an image of the sample S is constructed based on the calculated luminance value and displayed on the monitor 49. As a result, the user can observe the sample S while viewing the image displayed on the monitor 49.

  In this case, the PC 145 multiplies the number of photons by the reciprocal of the ratio of the actual exposure time to the reference exposure time, whereby the luminance value for image construction can be made constant regardless of the change in the number of photons. Then, by constructing an image of the sample S based on this luminance value, an image having a constant brightness can be acquired regardless of the fluorescence exposure time in the light receiving unit 42.

  As a result, according to the light detection system 140 and the microscope system 100 according to the present embodiment, the brightness of the displayed image is kept constant even when the exposure time is changed during the photon counting detection, and the sample S is obtained by a desired image. Can be observed. In addition, by the arithmetic processing, an appropriate luminance value can be determined flexibly according to the actual number of photons and exposure time, and an image of the sample S can be constructed with high accuracy.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to each of the above embodiments, and may be applied to embodiments in which these embodiments are appropriately combined, and is not particularly limited.

21 Scanner (scanning unit)
35 Objective lens 40,140 Photodetection system 41 Detector (photoelectric conversion unit)
42 light receiving unit 45 PC (image construction unit)
47 Input unit 55 Measuring instrument (photon measurement processing unit)
100 microscope system

Claims (4)

A photoelectric conversion unit that has a light receiving unit that receives observation light emitted from the sample, photoelectrically converts the observation light received by the light receiving unit, and outputs a current signal having a magnitude corresponding to the light amount;
A photon measurement processing unit for measuring the number of photons contained in the current signal output from the photoelectric conversion unit;
An input unit for inputting an exposure time during which the observation light is exposed in the light receiving unit;
Multiplied by the reciprocal of the ratio of the reference and made a predetermined the actual exposure time input by said input unit with respect to the reference exposure time is the exposure time actual exposure time, the number of photons measured by the photon counting processing unit And an image construction unit that constructs an image of the sample based on the luminance value obtained in this manner.   The image construction unit includes a plurality of lookup tables in which the number of photons and the luminance value are associated with each exposure time, and is measured by the photon measurement processing unit from the lookup table selected based on the actual exposure time. The light detection system according to claim 1, wherein the luminance value associated with the number of photons that has been read is read out. The image construction section, the number of photons measured by the photon counting processing unit, the actual exposure time and the light detection according to claim 1, with said reference exposure time by processing to calculate the luminance value system. The light detection system according to any one of claims 1 to 3,
A scanning unit that reflects illumination light emitted from a light source and scans the sample;
Irradiating the illumination light reflected by the scanning unit onto the sample, and an objective lens that collects the observation light emitted from the sample and received by the light receiving unit of the photoelectric conversion unit,
The microscope system which uses the scanning time of the said illumination light on the said sample by the said scanning part as said real exposure time.

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